Polycyclic aromatic hydrocarbons (PAH) are a class of ubiquitous environmental contaminants and are suspected to be a cause of cancer and toxicities of the immune and reproductive systems. It may be possible to reduce the incidence of cancer and toxicities caused by the exposure of susceptible individuals to PAHs by administering chemoprotective agents such as the prototype, oltipraz. One possible mechanism of action of oltipraz inhibition of PAH-induced cancer is induction of certain UDP-glucuronosyltransferases (UGTs), one of the major classes of Phase II detoxification enzymes. UGTs catalyze the conjugation of xenobiotic metabolites with UDP-glucuronic acid, resulting in decreased toxicity and increased excretion. The broad long-term objective of this project is to understand the mechanism of action of oltipraz chemoprotection against the PAH, benzo(a)pyrene (B(a)P).
The Specific Aims of this project are:
Specific Aim 1 : To show that oltipraz induces rat hepatic UGTs.
Specific Aim 2 : To demonstrate that oltipraz induces rat liver UGTs with activity towards 3-hydroxy-BP, BP-3,5-quinol, BP-7,8-diol, and other BP metabolites.
Specific Aim 3 : To determine if the UGT2B1 or UGT1.6 isoform represents a BP 7,8-diol UGT.
Specific Aim 4 : To determine whether oltipraz accelerates the cellular formation and excretion of BP glucuronides.
Specific Aim 5 : To demonstrate the cytoprotective roles of the oltipraz-responsive UGTs, UGT1.6 and UGT2B1, against BP.
Specific Aim 6 : To show that an AP-1-like element upstream of the UGT2B1 promoter is required for induction of rat UGT2B1 mRNA by oltipraz. To address these questions, the experimental approach will be to (1) pretreat rats with oltipraz, prepare microsomes from liver or other tissues, and analyze microsomal glucuronidating activities toward isoform-selective UGT substrates as well as the key toxic metabolites of BP; (2) identify oltipraz-responsive UGTs by analysis of induced UGT mRNAs; (3) determine if AHH-1 lymphoblast cells transfected with an UGT1.6 or UGT2B1 expression plasmid exhibit 7,8-diol UGT activity; (4) use high performance liquid chromatography to identify the products of BP metabolism in control and oltipraz-treated cells; (5) engineer lymphoblastic cells that express the UGT1.6 or EGT2B1 isoforms and compare the cells for sensitivity to cytotoxicity and mutagenicity of BP; and (6) use gene-reporter constructs to demonstrate oltipraz inducibility of UGT2B1 gene transcription. Biochemical methods include preparation of microsomes by differential centrifugation, assays of UGT activities by either spectrophotometric or radiometric ((14C)]-UDP-glucuronic acid) assays, RNA isolation and Northern analysis. Molecular methods include construction of UGT expression vectors, DNA transfections of AHH-1 (L3) cells by protoplast fusion, and hygromycin B selection of plasmid-bearing cells.
